Alzheimer's disease (AD) is a progressive disorder resulting in severe dementia which currently cannot be stopped. Abnormal accumulation of extracellular brain beta-amyloid is thought to cause AD and compounds that block beta-amyloid production are predicted to be effective for treating AD. Beta-amyloid is generated by cleavage from a larger protein, amyloid precursor protein (APR), by proteases called beta-secretases and gamma-secretases. Compounds that inhibit either of these secretases reduce beta-amyloid production and, therefore, are thought to be potentially effective AD drugs. Brain neurons secrete beta-amyloid by regulated and constitutive secretory pathways. We find that neurons secrete the vast majority of beta-amyloid via the regulated secretory pathway and that the cysteine proteases, cathepsin B and cathepsin L, are the beta- secretases of that pathway. Consequently, we find cathepsin B and cathepsin L are responsible for producing the majority of secreted beta-amyloid. Evidence for our finding is based on a wide range of experimental data. We purified beta-secretase activity from isolated regulated secretory vesicles and determined by microsequencing that the beta-secretase activity is due cathepsin B and cathepsin L. We found by immunoelectron microscopy that cathepsin B and cathepsin L are co-localized with APP and beta-amyloid in isolated secretory vesicles. We observed that cathepsin B and/or cathepsin L inhibitors block endogenous beta-secretase activity in isolated regulated secretory vesicles and stop the continued natural production of beta-amyloid that occurs after isolation of regulated secretory vesicles. Moreover, we found that cathepsin B and cathepsin L cleave with excellent efficiency the beta-secretase site found in wild-type APP, the APP form found in 99% of AD patients. Further, we found that a cathepsin B specific inhibitor, CA-074, dramatically reduces regulated secretion of Ab, but not its constitutive secretion, by neuronal chromaffin cells. Significantly, we show in this application that the cysteine protease inhibitor, e64d, reduces in vivo total brain and synaptosomal beta-amyloid in the guinea pig AD animal model. In this project, we will evaluate peptidomimetics that specifically inhibit cathepsin B or cathepsin L and which we find also inhibit beta-secretase activity in regulated secretory vesicles. Specifically, we will assess the affects of these peptidomimetics on beta-amyloid secretion in primary neuronal cultures and in vivo.